This invention relates to peptide fragments and analogs derived from the amino acid sequence of PBAN 1-33 NH2 (PBAN=pheromone biosynthesis activating neuropeptide) showing stimulatory and inhibitory biological activities at doses similar to those of PBAN 1-33 (10-100 pmol). These peptide fragments serve as a basis for the design of insecticides aimed at the disruption of pheromone production, melanization and other activities controlled by PBAN 1-33 NH2 and derived fragments.
Said invention further relates to a method for controlling adult female moths and insects by applying the above mentioned peptide fragments or peptidomimetic compounds derived from their sequence to any agricultural produce to inhibit pheromone production.
Neuropeptides play a key role in the regulation of a variety of physiological functions in insects. These endocrine cues are involved in embryonic and post-embryonic developmental processes (such as molting, diapause and metamorphosis), in homeostasis, osmoregulation, diuresis and digestion. Insect neuropeptides are also known to control essential behavioral patterns such as migration, mating and oviposition. In recent years, the development of improved chemical, biochemical and molecular techniques has facilitated the identification of insect neuropeptides and resulted in the isolation and characterization of over fifty compounds.
Due to their substantial involvement in a variety of physiological processes, studies of insect neuropeptides introduce a new insight into basic life processes which may serve as an excellent basis for comparative neuroendocrine studies aimed to understand the evolutionary history of basic biological events. In addition, insect neuropeptides provide new targets for pest control which are based on the interference with their activity. This strategy requires a better understanding of the cascade of events common to the neuroendocrine regulation, namely: biosynthesis, release, transport, binding and activation of the target organ. So far, our understanding of these events for most insect neuropeptides is very limited. The present invention concentrates centrates on some of these aspects comprising the activity of peptide fragments derived from PBAN, which is known to regulate sex-pheromone biosynthesis and melanization in moths.
Sexual communication between male and female moths is regulated mainly by sex pheromones. Sex pheromones are synthesized and secreted by the female moth from the pheromone gland which is located at the intersegmental membrane between the eighth and the ninth abdominal segments. The inability of the female moth to produce sex pheromones results in a marked decrease in mating and thus, a significant reduction in the size of the population. Due to their important role in mating, sex pheromones have been studied intensively. In the past 30 years more than two hundred sex pheromones were isolated and identified. These studies revealed that moths sex pheromones consist of blends of C10-C18 aliphatic compounds, most of which have one or more double bonds. The diversity between pheromones of different species is indicated by differences in the chain length, the position and configuration of the olefinic bonds, and by the chemical nature of the functional group. Most pheromones are aldehydes, alcohols or acetates. Some, however, may-appear as epoxides, ketones and hydrocarbons.
While the structural and behavioral aspects of sex pheromones in Lepidopterans have been studied intensively, the endogenous mechanisms that control sex pheromone biosynthesis are not fully understood. The possible involvement of a cerebral factor in this process was first proposed in 1971 by Riddiford and Williams (Biol. Bull. 140, 1-7). Direct evidence of the involvement of a neuroendocrine factor in the regulation of sex pheromone biosynthesis was demonstrated for the first time only thirteen years later by Raina and Klun, (Science 225, 531-533, 1984) in Helico-verpa zea. The neuroendocrine factor was termed pheromone biosynthesis activating neuropeptide (PBAN), and since 1984 its presence has been demonstrated in a variety of moth species. Since its discovery PBAN was characterized as a linear C-terminally amidated peptide containing 33 amino acids. The primary structure of PBAN has been fully identified in two moth species: Helicoverpa zea and Bombyx mori and its gene and cDNA have been cloned from the same two insects. PBAN is synthesized in the subesophageal ganglion and is transported to its target organ via the hemolymph and/or the ventral nerve cord. It is present in both male and female moth and its biological activity is mediated by cAMP and depends on the presence of Ca++ ions. In addition to its pheromonotropic activity PBAN and fragments derived from its sequence also control cuticular melanization, muscle contraction and diatause (for review see Altstein et al, Arch. Insect. Biochem. and Physiol. 22, 153-168, 1993; Raina, Ann. Rev. Entomol. 38, 329-349, 1993).
Despite the fact that PBAN has been studied for almost ten years, the understanding of its activity is quite limited, and most of the studies concentrate on: A) its release and transport; B) identification of its target organ; and C) its cellular activity. Among the various steps associated with the activity of the neuropeptide most effective and specific target for inhibition is the binding site of PBAN. Such an inhibition can be acquired by antagonists, which are selective inhibitors capable of locking the receptor site of the neuropeptide, and thus, preventing from the endogenous peptide to bind to the receptor and exert its biological activity. PBAN antagonists are, therefore, specific inhibitors for the production of sex pheromones. Since neuropeptides are chemically unstable and readily attacked by enzymes the strategy for the development of the antagonistic compounds is based on creating peptide-derived compounds (peptidomimetic compounds) which are biologically stable, and exhibit extended antagonistic activity. This strategy is novel and was never applied before for insect control.
In order to design peptidomimetic antagonists the structure-activity relationship of PBAN has to be revealed, and the active and inactive sequences in the molecule have to be identified. The main achievements of the study are:
A) The identification of the shortest fragment (six amino acids) derived from the sequence of Hez-PBAN that evokes pheromonotropic activity in the moth Heliothis peltigera at doses similar to those of Hez-PBAN 1-33 NH2 (synthetic PBAN, based on the sequence of the peptide isolated from Helicoveria zea).
B) The identification of a 10 amino acid fragment derived from the original sequence of PBAN which exhibits partial antagonistic activity.
The finding that these two fragments are short peptides introduces the following advantages:
A) The number of possible derivatives that has to be screened in the course of the search for a potent antagonist is several orders of magnitude lower than the number that has to be screened in the case of a large peptide. This may shorten significantly the first stage of the search for a lead antagonist.
B) The production of a 10 or less amino acid pedtide is based on available equipment and technologies and is most efficient.
C) This finding is in line with the common practice in the insecticide industry that insecticides, particularly those applied to adult insects, should be of low molecular weight, in order to penetrate the insect""s cuticle.
These achievements enable the application of strategies similar to those for rational drug design to search for peptidomimetic PBAN antagonists.
Two patent applications have been filed so far with respect to PBAN:
A) A Japanese patent application No. 4-208300 (Suzuki et. al) which relates to the full 33 amino acid sequence peptide isolated from silkworms, and also to some of its fragments. These peptides, according to this patent application are activating the pheromone bio-synthesis in silkworms. The Japanese application does not relate to the use of these peptides as a source and a basis for the design of lead compounds and potential agonists and antagonists for PRAN. Moreover, the experimental biological conditions and the concentrations in which the activity is observed in the J.P. patent application are totally different from the present invention. The concentrations of the peptides for obtaining activity are 100 times higher in the J.P. patent application than in the present application
B) A U.S. Pat. No. 5,032,576 (Raina et al.,) which relates to the isolation, characterization and synthesis of PBAN from Helicoverpa zea. The patent also refers to the use of the full length PBAN 1-33 NH2 and some of its analogs (all of which are 33 amino acid long) as methods for controlling female moths or larvae. Said US patent does not relate to the biological activity of shorter fragments derived from the sequence of Hez-PBAN or their use as means for insect control.
The present invention provides peptide fragments and analogs derived from the amino acid sequence of Hez-PBAN 1-33 NH2 showing stimulatory (agonistic) and inhibitory (antagonistic) biological activities at doses similar to those of PBAN 1-33 (10-100 pmol) for use as a basis for the design of insecticides aimed at the disruption of pheromone production, melanization and other activities controlled by PBAN 1-33 NH2 and derived fragments.
Said invention further provides a method for controlling adult female Heliothis, moths and insects by applying the above mentioned peptide fragments or peptidomimetic compounds derived from their sequence to any agricultural produce to inhibit pheromone production. More specifically, the preferred peptide fragments according to the invention are as follows:
a) R-Tyr-Phe-Ser-Pro-Arg-Leu-NH2 (SEQ ID NO: 2) wherein R represents H, Ac, Bz, Bzl, Et, showing stimulatory activity at doses of 10 pmol in a pheromonotropic bioassay at times ranging from 30-60 min.
b) H-Pro-Ala-Asp-Gln-Glu-Met-Tyr-Arg-Gln-Asp-COOH (SEQ ID NO: 3), showing inhibitory activity in the presence of PBAN 1-33 NH2.
c) H-Pro-Ala-Asp-Gln-Glu-Met-Tyr-Arg-Gln-Asp-COOH (SEQ ID NO:3) that shows inhibitory activity in the presence of PBAN 1-33 NH2 and or derived peptides resulting in the inhibition of pheromone production in female moths.
d) H-Pro-Ala-Asp-Gln-Glu-Met-Tyr-Arg-Gln-Asp-COOH (SEQ ID NO:3) that shows inhibitory activity in the presence of PBAN 1-33 NH2 and or derived peptides resulting in the inhibition of melanin production in moth larvae.